Immunogenic compositions comprising dal/dat double mutant, auxotrophic attenuated strains of Listeria and their methods of use

ABSTRACT

The invention includes auxotrophic attenuated mutants of  Listeria  and methods of their use as vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/660,194, filed Sep. 11, 2003, which is a continuation applicationof U.S. application Ser. No. 10/136,253, filed May 1, 2002, now U.S.Pat. No. 6,635,749, which is a divisional application of U.S.application Ser. No. 09/520,207, filed Mar. 7, 2000, now U.S. Pat. No.6,504,020, which is a divisional application of U.S. application Ser.No. 08/972,902, filed Nov. 18, 1997, now U.S. Pat. No. 6,099,848, whichare hereby incorporated by reference in their entirety.

GOVERNMENT INTEREST

This invention was supported in part by funds from the U.S. Government(NIH Grant Nos. AI-26919 and AI-27655) and the U.S. Government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to vaccine vectors comprising bacteria.

BACKGROUND OF THE INVENTION

The use of vaccines is a cost-effective medical tool for the managementof infectious diseases, including infectious diseases caused bybacteria, viruses, parasites, and fungi. In addition to effectingprotection against infectious diseases, vaccines may now also bedeveloped which stimulate the host's immune system to intervene in tumorgrowth.

Host immune responses include both the humoral immune response involvingantibody production and the cell-mediated immune response. Protectiveimmunization via vaccine has usually been designed to induce theformation of humoral antibodies directed against infectious agents,tumor cells, or the action of toxins. However, the control of certaindiseases characterized by the presence of tumor cells or by chronicinfection of cells with infectious agents, often requires acell-mediated immune response either in place of, or in addition to thegeneration of antibody. While the humoral immune response may be inducedusing live infectious agents and agents which have been inactivated, acellular immune response is most effectively induced through the use oflive agents as vaccines. Such live agents include live infectious agentswhich may gain access to the cytoplasm of host cells where the proteinsencoded by these agents are processed into epitopes which when presentedto the cellular immune system, induce a protective response.

Microorganisms, particularly Salmonella and Shigella which have beenattenuated using a variety of mechanisms, have been examined for theirability to encode and express heterologous antigens (Coynault et al.,1996, Mol. Microbiol. 22:149-160; Noriega et al., 1996, Infect. Immun.64:3055-3061; Brett et al., 1993, J. Immunol. 150:2869-2884; Fouts etal., 1995, Vaccine 13:1697-1705, Sizemore et al., 1995, Science270:299-302). Such bacteria may be useful as live attenuated bacterialvaccines which serve to induce a cellular immune response directedagainst a desired heterologous antigen.

Listeria monocytogenes (L. monocytogenes) is the prototypicintracellular bacterial pathogen which elicits a predominantly cellularimmune response when inoculated into an animal (Kaufmann, 1993, Ann.Rev. Immunol. 11:129-163). When used as a vector for the transmission ofgenes encoding heterologous antigens derived from infectious agents orderived from tumor cells, recombinant Listeria encoding and expressingan appropriate heterologous antigen have been shown to successfullyprotect mice against challenge by lymphocytic choriomeningitis virus(Shen et al., 1995, Proc. Natl. Acad. Sci. USA 92:3987-3991; Goossens etal., 1995, Int Immunol. 7:797-802) and influenza virus (Ikonomidis etal., 1997, Vaccine 15:433-440). Further, heterologous antigen expressingrecombinant Listeria have been used to protect mice against lethal tumorcell challenge (Pan et al., 1995, Nat. Med. 1:471-477; Paterson andIkonomidis, 1996, Curr. Opin. Immunol. 8:664-669). In addition, it isknown that a strong cell-mediated immune response directed against HIV-1gag protein may be induced in mice infected with a recombinant L.monocytogenes comprising HIV-1 gag (Frankel et al., 1995, J. Immunol.155:4775-4782).

Although the potential broad use of Listeria as a vaccine vector for theprevention and treatment of infectious disease and cancer hassignificant advantages over other vaccines, the issue of safety duringuse of Listeria is not trivial. The use of the most common strain ofListeria, L. monocytogenes, is accompanied by potentially severe sideeffects, including the development of listeriosis in the inoculatedanimal. This disease, which is normally food-borne, is characterized bymeningitis, septicemia, abortion and often a high rate of mortality ininfected individuals. While natural infections by L. monocytogenes arefairly rate and may be readily controlled by a number of antibiotics,the organism may nevertheless be a serious threat in immunocompromisedor pregnant patients. One large group individuals that might benefitfrom the use of L. monocytogenes as a vaccine vector are individuals whoare infected with HIV. However, because these individuals are severelyimmunocompromised as a result of their infection, the use of L.monocytogenes as a vaccine vector is undesirable unless the bacteria arefully and irreversibly attenuated.

There is a need for the development of a strain of L. monocytogenes foruse as a vaccine in and of itself and for use as a bacterial vaccinevector which is attenuated to the extent that it is unable to causedisease in an individual into whom it is inoculated. The presentinvention satisfies this need.

SUMMARY OF THE INVENTION

The invention includes a method of eliciting a T cell immune response toan antigen in a mammal comprising administering to the mammal anauxotrophic attenuated strain of Listeria which expresses the antigen,wherein the auxotrophic attenuated strain comprises a mutation in atleast one gene whose protein product is essential for growth of theListeria. In a preferred embodiment, the Listeria is L. monocytogenes.In another preferred embodiment, the auxotrophic attenuated strain isauxotrophic for the synthesis of D-alanine. In addition, the mutationcomprises a mutation in both the dal and the dat genes of the Listeria.

In one aspect of the invention, the auxotrophic attenuated strainfurther comprises DNA encoding a heterologous antigen, or theauxotrophic attenuated strain further comprises a vector comprising aDNA encoding a heterologous antigen.

The heterologous antigen may be an HIV-1 antigen.

The invention also includes a vaccine comprising an auxotrophicattenuated strain of Listeria which expresses an antigen, wherein theauxotrophic attenuated strain comprises a mutation in at least one genewhose protein product is essential for growth of the Listeria.

In preferred embodiments, the Listeria is L. monocytogenes. In otherpreferred embodiments, the auxotrophic attenuated strain is auxotrophicfor the synthesis of D-alanine. In yet other preferred embodiments, themutation comprises a mutation in both the dal and the dat genes of theListeria.

The auxotrophic attenuated strain may further comprise DNA encoding aheterologous antigen or a vector comprising a DNA encoding aheterologous antigen.

The heterologous antigen may be an HIV-1 antigen.

Also included in the invention is an isolated nucleic acid sequencecomprising a portion of a Listeria dal gene and an isolated nucleic acidsequence comprising a portion of a Listeria dat gene.

In addition, the invention includes an isolated strain of Listeriacomprising a mutation in a dal gene and a mutation in a dat gene whichrender the strain auxotrophic for D-alanine. In one aspect, the isolatedstrain of Listeria further comprises a heterologous antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the DNA sequence of the L. monocytogenes alanine racemase gene(dal) of L. monocytogenes and the amino acid sequence encoded thereby.The lysyl residue involved in the binding of pyridoxal-P is indicated byan asterisk.

FIG. 2 depicts the linear alignment of the deduced amino acid sequencesof the alanine racemases of L. monocytogenes (LMDAL), B.stearothermophilus, (BSTDAL), and B. subtilis (BSUBDAL). Identical aminoacids are boxed.

FIG. 3 is the DNA sequence of the L. monocytogenes D-amino acidaminotransferase gene (dat) and the amino acid sequence encoded thereby.The lysyl residue involved in the binding of pyridoxal-P is indicated byan asterisk.

FIG. 4 depicts the linear alignment of the deduced amino acid sequencesof the D-amino acid aminotransferases of L. monocytogenes (LMDAT), S.haemolyticus (SHAEDAT), B. sphaericus (BSPHDAT), and Bacillus speciesYM-1 (BSPDAT). Identical amino acids are boxed.

FIG. 5 is a graph depicting the growth requirement for D-alanine of thedal.sup.-dat.sup.-double mutant strain of L. monocytogenes. Thedal.sup.-dar.sup.-(daldat) and wild-type (L. monocytogenes+) strains ofL. monocytogenes were grown in liquid culture in BHI medium at37.degree. C. in the presence (+D-ala) or absence (−D-ala) of exogenousD-alanine (100 .mu.g/ml). In additional experiments, the mutant cellculture was also provided D-alanine after 30 minutes and after 60minutes.

FIG. 6 is a series of images of light micrographs depicting the growthof wild-type L. monocytogenes (Panel A) and the dal.sup.-dat.sup.-doublemutant strain of L. monocytogenes (Panel B) in J774 macrophages at 5hours after infection with about 5 bacteria per mouse cell. Panel Cillustrates an infection by double mutant bacteria in the continuouspresence of D-alanine (80 .mu.g/ml). Arrowheads point to some mutantbacteria.

FIG. 7 is a series of graphs depicting infection of mammalian cells withthe dal.sup.-dat.sup.-double mutant (open circles) and wild-type strainsof L. monocytogenes (closed circles). Mammalian cells which wereinfected included J774 murine macrophage-like cells (Panel A), primarymurine bone marrow macrophages (Panel B), and human epithelial cells(HeLa) (Panel C). Panel A also depicts mutant infection in the presenceof D-alanine (100 .mu.g/ml) (closed squares) and in the presence ofD-alanine from 0 to 4 hrs during infection (open squares).

FIG. 8 is a series of images of photomicrographs depicting theassociation of actin with intracytoplasmic wild-type L. monocytogenes(Panel A: 2 hours; Panel B: 5 hours) or with thedal.sup.-dat.sup.-double mutant of L. monocytogenes (Panel C: 2 hourswherein D-alanine was present from 0 to 30 minutes; Panel D: 5 hours,wherein D-alanine was present from 0 to 30 minutes; Panel E: 5 hours,wherein D-alanine was present continuously), following infection of J744cells with these bacteria. The images on the top row illustrate thebinding of FITC-labeled anti-Listerial antibodies to total bacteria,while the bottom row illustrates the binding of TRITC-labeled phalloidinto actin. The arrowheads point to some bacteria associated with actin.

FIG. 9 is a graph depicting the protection of BALB/c mice againstchallenge with ten times the LD.sub.50 of wild-type L. monocytogenes byimmunization with the dal.sup.-dat.sup.-double mutant strain of L.monocytogenes. Groups of 5 mice were immunized with the followingorganisms: (1) 4.times.10.sup.2 wild-type L. monocytogenes, (2)2.times.10.sup.7 dal.sup.-dal.sup.-(+D-alanine supplement), (3)2.times.10.sup.5 dal.sup.-dat.sup.-(+D-alanine supplement), (4)2.times.10.sup.4 dal.sup.-dat.sup.-(+D-alanine supplement), (5)2.times.10.sup.2 dal.sup.-dat.sup.-mutant dal.sup.-dat.sup.-(noD-alanine supplement). Mice were challenged 21-28 days afterimmunization. Log.sub.10 protection was calculated as described in theExamples.

FIG. 10 is a graph depicting the recovery of bacteria from spleens ofBALB/c mice following sublethal infection with wild type L.monocytogenes (closed circles), the dal.sup.-dat.sup.-mutant in theabsence of D-alanine (open circles), and the dal.sup.-dat.sup.-mutant inthe presence of 20 mg D-alanine (open squares). The points at day 0illustrate the total number of organisms injected, not the number ofbacteria per spleen.

FIG. 11 is a series of graphs depicting the cytolytic activity ofsplenocytes isolated from mice at 10-14 days after infection with inFIG. 11A, wild type L. monocytogenes (.circle-solid.largecircle.), ornaive control (.box-solid.quadrature.). FIG. 11B,dal.sup.-dat.sup.-mutant: 3.times.10.sup.7 bacteria (.DELTA.);3.times.10.sup.7 bacteria with boost at 10 days (.DELTA.);3.times.10.sup.7 bacteria wherein animals were provided D-alaninesubcutaneously (.circle-solid.largecircle.); 3.times.10.sup.7 bacteriaplus 2 mg/ml D-alanine (.box-solid.) or 0.2 mg/ml D-alanine in drinkingwater (.tangle-solidup.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vaccines comprising attenuated strainsof Listeria, wherein the bacteria have been attenuated by theintroduction of auxotrophic mutations in the Listeria genomic DNA. Thesestrains are herein referred to as attenuated auxotrophic strains or “AAstrains” of Listeria.

It has been discovered in the present invention that the administrationof an AA strain of Listeria to a mammal results in the development of ahost cytotoxic T cell (CTL) response directed against Listeria followingsurvival of the AA strain in the mammal for a time sufficient for thedevelopment of the response. The AA strain provides protection againstchallenge by L. monocytogene and is therefore suitable for use in avaccine for protection against infection by this organism. The AA strainof the invention may thus be employed as a vaccine for the preventionand/or treatment of infection by Listeria. In addition, the AA strain ofthe invention may have added to it a heterologous gene wherein the geneis expressed by the AA strain. Such AA strains encoding additionalheterologous genes are useful as bacterial vector vaccines for theprevention and/or treatment of infection caused by any number ofinfectious agents and for the prevention and/or treatment of tumors inmammals.

AA strains of Listeria that are auxotrophic for D-alanine arecontemplated as part of this invention.

By the term “auxotrophic for D-alanine”, as used herein, is meant thatthe AA strain of Listeria is unable to synthesize D-alanine in that itcannot grow in the absence of D-alanine and therefore requiresexogenously added D-alanine for growth.

D-alanine is required for the synthesis of the peptidoglycan componentof the cell wall of virtually all bacteria, and is found almostexclusively in the microbial world. Wild-type Listeria speciessynthesize D-alanine and thus do not require exogenously added D-alaninefor growth. An AA strain of L. monocytogenes has been discovered in thepresent invention which is unable to synthesize D-alanine. This organismmay be grown in the laboratory but is incapable of growth outside thelaboratory in unsupplemented environments, including in the cytoplasm ofeukaryotic host cells, the natural habitat of this organisms duringinfection. Such strains of Listeria are useful as vaccines.

By the term “vaccine,” as used herein, is meant a population of bacteriawhich when inoculated into a mammal has the effect of stimulating acellular immune response comprising a T cell response. The T cellresponse may be a cytotoxic T cell response directed againstmacromolecules produced by the bacteria. However, the induction of a Tcell response comprising other types of T cells by the vaccine of theinvention is also contemplated. For example, Listeria infection alsoinduces both CD4+ T cells and CD8+ T cells. Induced CD4+ T cells areresponsible for the synthesis of cytokines, such as interferon-.gamma.,IL-2 and TNF-.alpha. CD8+ T cells may be cytotoxic T cells and alsosecrete cytokines such as interferon-.gamma. and TNF-.alpha. All ofthese cells and the molecules synthesized therein play a role in theinfection and subsequent protection of the host against Listeria.Cytokines produced by these cells activate additional T cells and alsomacrophages and recruit polymorphonuclear leukocytes to the site ofinfection.

Both prophylactic and therapeutic vaccines are contemplated as beingwithin the scope of the present invention, that is, vaccines which areadministered either prior to or subsequent to the onset of disease areincluded in the invention.

D-alanine auxotrophic mutants useful as vaccine vectors may be generatedin a number of ways. As described in the Examples presented herein,disruption of one of the alanine racemase gene (dal) or the D-amino acidaminotransferase gene (dat), each of which is involved in D-alaninesynthesis, did not result in a bacterial strain which requiredexogenously added D-alanine for growth. However, disruption of both thedal gene and the dat gene generated an AA strain of Listeria whichrequired exogenously added D-alanine for growth.

The generation of AA strains of Listeria deficient in D-alaninesynthesis may be accomplished in a number of ways that are well known tothose of skill in the art, including deletion mutagenesis, insertionmutagenesis, and mutagenesis which results in the generation offrameshift mutations, mutations which effect premature termination of aprotein, or mutation of regulatory sequences which affect geneexpression. Mutagenesis can be accomplished using recombinant DNAtechniques or using traditional mutagenesis technology using mutagenicchemicals or radiation and subsequent selection of mutants. Deletionmutants are preferred because of the accompanying low probability ofreversion of the auxotrophic phenotype. Mutants of D-alanine which aregenerated according to the protocols presented herein may be tested forthe ability to grow in the absence of D-alanine in a simple laboratoryculture assay. Those mutants which are unable to grow in the absence ofthis compound are selected for further study.

In addition to the aforementioned D-alanine associated genes, othergenes involved in D-alanine synthesis may be used as targets formutagenesis of Listeria. Such genes include, but are not limited to anyother known or heretofore unknown D-alanine associated genes.

Genes which are involved in the synthesis of other metabolic componentsin a bacterial cell may also be useful targets for the generation ofattenuated auxotrophic mutants of Listeria, which mutants may also becapable of serving as bacterial vaccine vectors for use in the methodsof the present invention. The generation and characterization of suchother AA strains of Listeria may be accomplished in a manner similar tothat described herein for the generation of D-alanine deficient AAstrains of Listeria.

Additional potential useful targets for the generation of additionalauxotrophic strains of Listeria include the genes involved in thesynthesis of the cell wall component D-glutamic acid. To generateD-glutamic acid auxotrophic mutants, it is necessary to inactivate thedat gene, which is involved in the conversion of D-glu+pyr toalpha-ketoglutarate+D-ala and the reverse reaction. It is also necessaryto inactivate the glutamate racemase gene, dga. Other potential usefultargets for the generation of additional auxotrophic strains of Listeriaare the genes involved in the synthesis of diamimopimelic acid. In thisinstance, a gene encoding aspartate beta-semialdehyde dehydrogenase maybe inactivated (Sizemore et al., 1995, Science 270:299-302).

By the term “attenuation,” as used herein, is meant a diminution in theability of the bacterium to cause disease in an animal. In other words,the pathogenic characteristics of the attenuated Listeria strain havebeen lessened compared with wild-type Listeria, although the attenuatedListeria is capable of growth and maintenance in culture. Using as anexample the intravenous inoculation of Balb/c mice with an attenuatedListeria, the lethal dose at which 50% of inoculated animals survive(LD.sub.50) is preferably increased above the LD.sub.50 of wild-typeListeria by at least about 10-fold, more preferably by at least about100-fold, more preferably at least about 1,000 fold, even morepreferably at least about 10,000 fold, and most preferably at leastabout 100.000-fold. An attenuated strain of Listeria is thus one whichdoes not kill an animal to which it is administered, or is one whichkills the animal only when the number of bacteria administered is vastlygreater than the number of wild type non-attenuated bacteria which wouldbe required to kill the same animal. An attenuated bacterium should alsobe construed to mean one which is incapable of replication in thegeneral environment because the nutrient required for its growth is notpresent therein. Thus, the bacterium is limited to replication in acontrolled environment wherein the required nutrient is provided. Theattenuated strains of the present invention are thereforeenvironmentally safe in that they are incapable of uncontrolledreplication.

It is believed that any Listeria species capable of infectious diseasemay be genetically attenuated according to the methods of the presentinvention to yield a useful and safe bacterial vaccine, provided theattenuated Listeria species exhibits an LD.sub.50 in a host organismthat is significantly greater than that of the non-attenuated wild typespecies. Thus, strains of Listeria other than L. monocytogenes may beused for the generation of attenuated mutants for use as vaccines.Preferably, the Listeria strain useful for the generation of attenuatedvaccines is L. monocytogenes.

An AA strain of Listeria may be generated which encodes and expresses aheterologous antigen. The heterologous antigen encoded by the AA strainof Listeria is one which when expressed by Listeria is capable ofproviding protection in an animal against challenge by the infectiousagent from which the heterologous antigen was derived, or which iscapable of affecting tumor growth and metastasis in a manner which is ofbenefit to a host organism. Heterologous antigens which may beintroduced into an AA strain of Listeria by way of DNA encoding the samethus include any antigen which when expressed by Listeria serves toelicit a cellular immune response which is of benefit to the host inwhich the response is induced. Heterologous antigens therefore includethose specified by infectious agents, wherein an immune responsedirected against the antigen serves to prevent or treat disease causedby the agent. Such heterologous antigens include, but are not limitedto, viral, bacterial, fungal or parasite surface proteins and any otherproteins, glycoproteins, lipoprotein, glycolipids, and the like.Heterologous antigens also include those which provide benefit to a hostorganism which is at risk for acquiring or which is diagnosed as havinga tumor. The host organism is preferably a mammal and most preferably,is a human.

By the term “heterologous antigen,” as used herein, is meant a proteinor peptide, a glycoprotein or glycopeptide, a lipoprotein orlipopeptide, or any other macromolecule which is not normally expressedin Listeria, which substantially corresponds to the sane antigen in aninfectious agent, a tumor cell or a tumor-related protein. Theheterologous antigen is expressed by an AA strain of Listeria, and isprocessed and presented to cytotoxic T-cells upon infection of mammaliancells by the AA strain. The heterologous antigen expressed by Listeriaspecies need not precisely match the corresponding unmodified antigen orprotein in the tumor cell or infectious agent so long as it results in aT-cell response that recognizes the unmodified antigen or protein whichis naturally expressed in the mammal.

By the term “tumor-related antigen,” as used herein, is meant an antigenwhich affects tumor growth or metastasis in a host organism. Thetumor-related antigen may be an antigen expressed by a tumor cell, or itmay be an antigen which is expressed by a non-tumor cell, but which whenso expressed, promotes the growth or metastasis of tumor cells.

The types of tumor antigens and tumor-related antigens which may beintroduced into Listeria by way of incorporating DNA encoding the same,include any known or heretofore unknown tumor antigen.

The heterologous antigen useful in vaccine development may be selectedusing knowledge available to the skilled artisan, and many antigenicproteins which are expressed by tumor cells or which affect tumor growthor metastasis or which are expressed by infectious agents are currentlyknown. For example, viral antigens which may be considered as useful asheterologous antigens include but are not limited to the nucleoprotein(NP) of influenza virus and the gag protein of HIV. Other heterologousantigens include, but are not limited to, HIV env protein or itscomponent parts gp120 and gp41, HIV nef protein, and the HIV polproteins, reverse transcriptase and protease. In addition, other viralantigens such as herpesvirus proteins may be useful. The heterologousantigens need not be limited to being of viral origin. Parasiticantigens, such as, for example, material antigens, are included, as arefungal antigens, bacterial antigens and tumor antigens.

As noted herein, a number of proteins expressed by tumor cells are alsoknown and should be included in the list of heterologous antigens whichmay be inserted into the vaccine strain of the invention. These include,but are not limited to, the bcr/abl antigen in leukemia, HPVE6 and E7antigens of the oncogenic virus associated with cervical cancer, theMAGE1 and MZ2-E antigens in or associated with melanoma, and the MVC-1and HER-2 antigens in or associated with breast cancer.

The introduction of DNA encoding a heterologous antigen into a strain ofListeria may be accomplished, for example, by the creation of arecombinant Listeria in which DNA encoding the heterologous antigen isharbored on a vector, such as a plasmid for example, which plasmid ismaintained and expressed in the Listeria species. Alternatively, DNAencoding the heterologous antigen may be stably integrated into theListeria chromosome by employing, for example, transposon mutagenesis orby homologous recombination. A preferred method for producingrecombinant Listeria having a gene encoding a heterologous antigenintegrated into the chromosome thereof, is the induction of homologousrecombination between a temperature sensitive plasmid comprising DNAencoding the antigen and Listeria chromosomal DNA. Stable transformantsof Listeria which express the desired antigen may be isolated andcharacterized as described herein in the experimental examples. Thismethod of homologous recombination is advantageous in that site directedinsertion of DNA encoding the heterologous antigen is effected, therebyminimizing the possibility of disruption of other areas of the Listeriachromosome which may be essential for growth of this organism.

Several approaches may be employed to express the heterologous antigenin Listeria species as will be understood by one skilled in the art oncearmed with the present disclosure. Genes encoding heterologous antigensare preferably designed to either facilitate secretion of theheterologous antigen from the bacterium or to facilitate expression ofthe heterologous antigen on the Listeria cell surface.

While the heterologous antigen preferably comprises only a desiredantigen along with appropriate signal sequences and the like, alsocontemplated in the invention is a fusion protein which comprises thedesired heterologous antigen and a secreted or cell surface protein ofListeria. Listeria proteins which are suitable components of such fusionproteins include, but are not limited to, listeriolysin O (LLO) andphosphatidylinositol-specific phospholipase (PI-PLC). A fusion proteinmay be generated by ligating the genes which encode each of thecomponents of the desired fusion protein, such that both genes are inframe with each other. Thus, expression of the ligated genes results ina protein comprising both the heterologous antigen and the listerialprotein. Expression of the ligated genes may be placed under thetranscriptional control of a listerial promoter/regulatory sequence suchthat expression of the gene is effected during growth and replication ofthe organism. Signal sequences for cell surface expression and/orsecretion of the fused protein may also be added to genes encodingheterologous antigens in order to effect cell surface expression and/orsecretion of the fused protein.

When the heterologous antigen is used alone (i.e., in the absence offused Listeria sequences), it may be advantageous to fuse thereto signalsequences for cell surface expression and/or secretion of theheterologous antigen. The procedures for accomplishing this are wellknow in the art of bacteriology and molecular biology.

The DNA encoding the heterologous antigen which is expressed in thevaccine strain of the invention must be preceeded by a suitable promoterto facilitate such expression. The appropriate promoter/regulatory andsignal sequences to be used will depend on the type of listerial proteindesired in the fusion protein and will be readily apparent to thoseskilled in the art of listeria molecular biology. For example, preferredL. monocytogenes promoter/regulatory and/or signal sequences which maybe used to direct expression of a fusion protein include, but are notlimited to, sequences derived from the Listeria hly gene which encodesLLO, the Listeria p60 gene (Bouwer et al., 1996, Infect. Immun.64:2515-2522) and possibly the Listeria actA gene which encodes asurface protein necessary for L. monocytogenes actin assembly. Otherpromoter sequences which might be useful in some circumstances includethe plcA gene which encodes PI-PLC, the listeria mpl gene, which encodesa metalloprotease, the listeria plcB gene encoding a phospholipase C,and the listeria inlA gene which encodes internalin, a listeria membraneprotein. For a review of genes involved in L. monocytogenespathogenesis, see Portnoy et al. (1992, Infect. and Immun.60:1263-1267). It is also contemplated as part of this invention thatheterologous regulatory elements such as promoters derived from phageand promoters or signal sequences derived from other bacterial speciesmay be employed for the expression of a heterologous antigen by theListeria species.

Examples of the use of recombinant L. monocytogenes strains that expressa heterologous antigen for induction of an immune response against tumorcell antigens or infectious agent antigens are described in U.S. patentapplication Ser. Nos. 08/366,372 and 08/366,477, respectively. Thedisclosures of these two patent applications are hereby incorporatedherein by reference.

The data presented herein indicate that certain AA strains of Listeriamay undergo osmotic lysis following infection of a host cell. Thus, ifthe Listeria which is introduced into the host cell comprises a vector,the vector is released into the cytoplasm of the host cell. The vectormay comprise DNA encoding a heterologous antigen. Uptake into thenucleus of the vector DNA enables transcription of the DNA encoding theheterologous antigen and subsequent expression of the antigen in and/orsecretion of the same from the infected host cell. Typically, the vectoris a plasmid that is capable of replication in Listeria. The vector mayencode a heterologous antigen, wherein expression of the antigen isunder the control of eukaryotic promoter/regulatory sequences. Typicalplasmids having suitable promoters that might be employed include, butare not limited to, pCMVbeta comprising the immediate earlypromoter/enhancer region of human cytomegalovirus, and those whichinclude the SV40 early promoter region or the mouse mammary tumor virusLTR promoter region.

Thus, it is also contemplated as part of the present invention that AAstrains of Listeria may be employed as a vaccine for the purpose ofstimulating a CTL immune response against an infectious agent or a tumorcell, wherein the AA strain comprises a vector encoding a heterologousantigen that may be expressed using a eukaryotic expression system.According to the invention, the vector is propagated in the AA strain ofListeria concomitant with the propagation of the AA strain itself. Thevector may be, for example, a plasmid that is capable of replication inthe AA strain or the vector may be lysogenic phage. The vector mustcontain a prokaryotic origin of replication and must not contain aeukaryotic origin of replication in order that the vactor is capable ofreplication in a prokaryotic cell but, for safety reasons, is renderedabsolutely incapable of replication in eukaryotic cells.

A cytotoxic T-cell response in a mammal is defined as the generation ofcytotoxic T-cells capable of detectably lysing cells presenting anantigen against which the T cell response is directed. Preferably,within the context of the present invention, the T cell response isdirected against a heterologous antigen expressed in an AA strain ofListeria or which is expressed by a vector which is delivered to a cellvia Listeria infection. Assays for a cytotoxic T-cell response are wellknown in the art and include, for example, a chromium release assay(Frankel et al., 1995, J. Immunol. 155:4775-4782). In addition to achromium release assay, an assay for released lactic acid dehydrogenasemay be performed using a Cytotox 96 kit obtained from Promega Biotech,WI.

In preferred embodiments and using a chromium release assay, at aneffector cell to target cell ratio of about 50:1, the percentage oftarget cell lysis is preferably at least about 10% above the backgroundlevel of cell lysis. The background level of cell lysis is the percentlysis of cells which do not express the target antigen. More preferably,the percentage of target cell lysis is at least about 20% abovebackground; more preferably, at least about 40% above background; morepreferably, at least about 60% above background; and most preferably, atleast about 70% above background.

The vaccines of the present invention may be administered to a hostvertebrate animal, preferably a mammal, and more preferably a human,either alone or in combination with a pharmaceutically acceptablecarrier. The vaccine is administered in an amount effective to induce animmune response to the Listeria strain itself or to a heterologousantigen which the Listeria species has been modified to express. Theamount of vaccine to be administered may be routinely determined by oneof skill in the art when in possession of the present disclosure. Apharmaceutically acceptable carrier may include, but is not limited to,sterile distilled water, saline, phosphate buffered solutions orbicarbonate buffered solutions. The pharmaceutically acceptable carrierselected and the amount of carrier to be used will depend upon severalfactors including the mode of administration, the strain of Listeria andthe age and disease state of the vaccinee. Administration of the vaccinemay be by an oral route, or it may be parenteral, intranasal,intramuscular, intravascular, intrarectal, intraperitoneal, or any oneof a variety of well-known routes of administration. The route ofadministration may be selected in accordance with the type of infectiousagent or tumor to be treated.

The vaccines of the present invention may be administered in the form ofelixirs, capsules or suspensions for oral administration or in sterileliquids for parenteral or intravascular administration. The vaccine mayalso be administered in conjunction with a suitable adjuvant, whichadjuvant will be readily apparent to the skilled artisan.

The immunogenicity of the AA strain of the invention may be enhanced inseveral ways. For example, a booster inoculation following the initialinoculation may be used to induce an enhanced CTL response directedagainst the AA strain.

In another approach, transient suppression of the auxotrophic phenotypeof the AA strain is accomplished by providing the AA strain with therequired nutrient for a period of time shortly before, after, orconcomitant with administration of the Listeria vaccine to the host. Theorganism will replicate for the brief period during which the nutrientis present, after which, upon exhaustion of the supply of the nutrient,the organism will cease replication. This brief period of controlledreplication will serve to provide more organisms in the host in a mannersimilar to that of natural infection by Listeria, which should stimulatean enhanced CTL response directed against the organism and antigensexpressed thereby.

In yet another approach, the use of a suicide plasmid may be employed toconditionally suppress the attenuation of the Listeria AA strain bytemporarily supplying the missing enzyme or enzymes to the bacterium forsynthesis of the essential nutrient. A suitable suicide plasmid includespKSV7, the same plasmid which was used to mediate insertion of genesinto the Listeria chromosome as described herein. This plasmid containsa gram positive (for use in Listeria), temperature-sensitive replicationsystem such that growth at 37-40.degree. C. inhibits plasmid replicationin 25 Listeria. This plasmid also contains an E. coli replication systemwhich is not temperature-sensitive (Smith et al., 1992, Biochimie74:705-711). The plasmid, or even more temperature-sensitive derivativesthereof, may be further modified by inserting an alanine racemase geneinto the plasmid, which modified plasmid is then inserted into an AAstrain of Listeria. Listeria cells having the plasmid inserted therein,are replicated at 30.degree. C. for a short period of time in order thatsome molecules of racemase are accumulated in the cytoplasm. TheListeria cells, so replicated are then injected into an animal or ahuman, wherein plasmid replication then ceases because of thetemperature sensitive nature of the replication system at 37.degree. C.Essentially, the cells would divide only a few times until the availableracemase becomes diluted out, wherein the cells would cease replicationaltogether and become attenuated again. To ensure even tightertemperature sensitive replication, a temperature sensitive promoter maybe used to regulate expression of the racemase gene and/or temperaturesensitive mutations may be created in the racemase gene itself.

For treatment of cancer, the vaccine of the invention may be used toprotect people at high risk for cancer. In addition, the vaccine may beused as an immunotherapeutic agent for the treatment of cancer followingdebulking of tumor growth by surgery, conventional chemotherapy, orradiation treatment. Patients receiving such treatment may beadministered a vaccine which expresses a desired tumor antigen for thepurpose of generating a CTL response against any residual tumor cells inthe individual. The vaccine of the present invention may also be used toinhibit the growth of any previously established tumors in a human byeither eliciting a CTL response directed against the tumor cells per se,or by eliciting a CTL response against cells which synthesize tumorpromoting factors, wherein such a CTL response serves to kill thosecells thereby diminishing or ablating the growth of the tumor.

The vaccine of the invention may be maintained in storage until use.Storage may comprise freezing the vaccine, or maintaining the vaccine at4.degree. C., room temperature, or the vaccine may first be lyophilizedand then stored.

The invention particularly contemplates administration of a vaccine to ahuman for the purpose of preventing, alleviating, or ablating HIVinfection. The protocol which is described herein for the administrationof a vaccine to a human for the purpose of treating HIV infection isprovided as an example of how to administer an attenuated auxotrophicListeria strain as a vaccine to a human. This protocol should not beconstrued as being the only protocol which can be used, but rather,should be construed merely as an example of the same. Other protocolswill become apparent to those skilled in the art when in possession ofthe present invention.

Essentially, an auxotrophic strain of L. monocytogenes which requiresD-alanine for growth is constructed as described in the examples. Themutant is constructed by generating deletion mutations in both the dalgene and the dat gene, essentially following the procedures of Camilliet al., (1993, Mol. Microbiol. 8:143-157). The mutant strain is thenmodified using recombinant DNA techniques to express an HIV-1 antigen,preferably an antigenic portion: of the gag protein, essentially asdescribed in Frankel et al. (1995, J. Immunol. 155:4775-4778). A humanis then immunized by injecting a solution containing the auxotrophic L.monocytogenes strain and a supplement of D-alanine.

One of ordinary skill in the art will know the quantities of cells andD-alanine which should be administered to the human based upon aknowledge of the dosages provided herein which are administered to mice.For example, in BALB/c mice, 10.sup.7 cells and 20 mg of D-alanine arethe preferred dosages. Subsequent injections of the modified L.monocytogenes cells and D-alanine may also be given to boost the immuneresponse.

Other HIV-1 antigens or proteins that may be used to generate a vaccinein accordance with this invention are the HIV env protein or itscomponent parts, gp120 and gp 41, HIV gag, HIV nef and HIV pol or itscomponent parts, reverse transcriptase and protease.

Isolated nucleic acid sequences encoding the dal gene and the dat geneof L. monocytogenes are also contemplated as part of this invention. Inaddition to their utility in generating deletion mutants of L.monocytogenes as disclosed herein, these isolated nucleic acid sequencesencoding the dal gene and the dat gene may be used as probes and primersin identifying homologous genes in other Listeria species using PCR andother hybridization technology available in the art and described, forexample, in Sambrook, et al. (1989, In: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York; Innis et al., ed.,1990, In: PCR Protocols, Academic Press, Inc., San Diego). Additionally,the isolated nucleic acid sequences encoding dal or dat may be used toconstruct a suicide plasmid that expresses one or both of the genes. Thesuicide plasmid(s) may be used to complement the D-alanine Listeriaauxotrophs for a limited time after immunization as disclosed herein.

An “isolated nucleic acid”, as used herein, refers to a nucleic acidsequence, a DNA or an RNA or fragment thereof which has been separatedfrom the sequences which flank it in a naturally occurring state, e.g. aDNA fragment which has been removed from the sequences which arenormally adjacent to the fragment, e.g., the sequences adjacent to thefragment in a genome in which it naturally occurs. The term also appliesto nucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNAor proteins, which naturally accompany it in the cell. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote; or which exists as aseparate molecule (e.g, as a cDNA or a genomic or cDNA fragment producedby PCR amplification, restriction enzyme digestion or chemicalsynthesis) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

Typically probes and primers for use in identifying other dal and datgenes will comprise a portion of a Listeria dal or dat gene that is atleast about 15 consecutive nucleotides. More typically, a probe orprimer comprises a portion of at least about 20, even more typically, atleast about 30 and even more typically, at least about 40 consecutivenucleotides of a dal or dat gene of Listeria.

In other related aspects, the invention includes a vectors whichcomprises an isolated nucleic acid encoding dal or dat and which ispreferably capable of directing expression of the protein encoded by thenucleic acid in a vector-containing cell. The invention further includescells comprising a vector encoding dal or dat, including bothprokaryotic and eukaryotic cells.

The isolated nucleic acids of the invention should be construed toinclude an RNA or a DNA sequence specifying the dal gene or the datgene, and any modified forms thereof, including chemical modificationsof the DNA or RNA which render the nucleotide sequence more stable whenit is cell free or when it is associated with a cell. Chemicalmodifications of nucleotides may also be used to enhance the efficiencywith which a nucleotide sequence is taken up by a cell or the efficiencywith which it is expressed in a cell. Any and all combinations ofmodifications of the nucleotide sequences are contemplated in thepresent invention.

The invention should not be construed as being limited solely to the DNAand amino acid sequences shown in FIGS. 1 and 3. Once armed with thepresent invention, it is readily apparent to one skilled in the art thatany other DNA and encoded amino acid sequence of the dal and dat genesof other Listeria species may be obtained by following the proceduresdescribed herein. The invention should therefore be construed to includeany and all dal and dat DNA sequence and corresponding amino acidsequence, having substantial homology to the dal and dat DNA sequence,and the corresponding amino acid sequence, shown in FIGS. 1 and 3,respectively. Preferably, DNA which is substantially homologous is about50% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous and most preferably about 90% homologousto the dal or dat DNA sequence shown in FIGS. 1 and 3, respectively.Preferably, an amino acid sequence which is substantially homologous isabout 50% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous and most preferably about 90% homologousto the amino acid sequences encoded by the dal and dat genes shown inFIGS. 1 and 3, respectively.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGCG5′ share 50%homology.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

The experimental examples described herein provide procedures andresults which establish that attenuated auxotrophic mutants of L.monocytogenes are useful as vaccines for eliciting a CTL response.

Materials and Methods useful in the construction and use of anattenuated auxotrophic L. monocytogenes strain are now described.

Bacteria and plasmids. The L. monocytogenes strain 10403S (Portnoy etal., 1988, supra) comprises the wild-type organism used in thesestudies. This organism was propagated in brain/heart infusion medium(BHI) (Difco Labs). L. monocytogenes strain 10403S has an LD.sub.50 ofapproximately 3.times.10.sup.4 when injected intravenously orintraperitoneally into BALB/c mice (Schafer et al., 1992, J. Immunol.149:53-59).

E. coli DH5a was used for cloning. This organism was propagated in Luriabroth (Sambrook et al., 1989, supra). The plasmid pKSV7, which was usedfor allelic exchange reactions in L. monocytogenes, is a shuttle vectorcapable of replication in E. Coli, where it is selected in the presenceof 50 .mu.g of ampicillin per ml of media, and in L. monocytogenes,wherein replication of the plasmid is temperature sensitive and isselected in the presence of 10 .mu.g of chloramphenicol per ml of media(Smith et al., 1992, Biochimie 74:705-711). Plasmid DNA obtained from E.coli and total DNA (chromosomal and plasmid) from obtained from Listeriamonocytogenes were isolated using standard methods (Sambrook et al.,1989, supra).

Identification of D-alanine synthesis genes in L. monocytogenes byhomology with D-alanine synthesis genes in other gram positiveorganisms. Based on sequences of the alanine racemase gene (dal) ingram-positive organisms (Ferrari et al., 1985, Bio/technology3:1003-1007; Tanizawa et al., 1988, Biochemistry 27:1311-1316), primerswere designed which corresponded to two 20 base consensus sequences fromhighly conserved regions at the 5′ and 3′ ends of the dal gene. Theseprimers were modified to reflect the preferred codon usage in Listeria.These primers were used in a PCR reaction using chromosomal DNA fromeither L. monocytogenes or B. subtilis as templates. A similar sized PCRproduct (850 nucleotides) was obtained from both L. monocytogenes and B.subtilis. Analysis of the 850 nucleotide PCR product from the Listeriatemplate, and the amino acid sequence encoded thereby, indicatedsubstantial homology with the alanine racemase genes of the othergram-positive organisms.

A similar strategy was used to identify and sequence a portion of aD-amino acid aminotransferase gene (dat) of Listeria, based on sequencesin B. sphaericus, B. species YM-1 (Tanizawa et al., 1989, supra), andPucci et al., 1995, J. Bacteriol. 177:336-342). Primers based on datsequence in the other gram positive organisms was used for PCRamplification of L. monocytogenes DNA and a PCR product of about 400nucleotides was obtained. Analysis of the DNA sequence of the 400nucleotide PCR product, and the amino acid sequence encoded thereby,indicated substantial homology with the aminotransferase genes of theother gram positive organisms.

Strategy for sequence determination of the complete genes. The sequenceof the remaining portions of the L. monocytogenes dal gene adjoined tothe 5′ and 3′ ends of the central PCR product was determined usinganchored PCR reactions (Rubin et al., 1993, Proc. Natl. Acad. Sci. USA90:9280-9284). Briefly, this procedure utilized a BglII-restrictiondigest (for the 5′ portion of the gene) or a XbaI digest (for the 3′portion of the gene) of Listeria chromosomal DNA. The ends of thedigested Listeria chromosomal DNA were then ligated to a small fragmentof DNA containing the T7 promoter. A 5′-portion PCR product and a3′-portion PCR product were then made and sequenced using primers fromwithin the central dal gene PCR product and a second primer homologousto the T7 promoter fragment. This procedure permitted determination ofthe entire sequence of the dal gene.

The sequence of the remainder of the dat gene was determined by use ofan inverse PCR reaction (Collins et al., 1984, Proc. Natl. Acad. Sci.USA 81:6812-6816; Triglia et al., 1988, Nucl. Acids Res. 16:8186).Briefly, a HindIII digest of Listeria chromosomal DNA was permitted toself-ligate under conditions of low DNA concentration so that mainlysingle circular molecules would form. Outward-directing primers withhomologies at the two ends of the original PCR segment of the gene werethen used to make a new PCR product that began at the 5′-end of theoriginal PCR segment, proceeded to the 5′-end of the gene through theHindIII self-ligation site and into the 3′-end of the gene. Using thismethod, the entire dat gene sequence was obtained.

Production of mutations in Listeria dal and dat genes. The dal gene wasinactivated by means of a double allelic exchange reaction following theprotocol of Camilli et al. (Camilli et al., 1993, Mol. Microbiol.8:143-157). A is shuttle plasmid pKSV7 (Smith et al., 1992, supra)construct containing an erythromycin gene (Shaw and Clewell, 1985, J.Bacteriol. 164:782-796) situated between a 450-base pair fragment of the5′ end of the 850-base pair dal gene PCR product and a 450-base pairfragment of the 3′ end of the dal gene PCR product was introduced intoListeria to produce a double allelic exchange reaction between thechromosomal dal gene and the plasmid pKSV7 dal construct. A dal deletionmutant covering about 25% of the gene in the region of its active sitewas obtained.

The chromosomal dat gene of L. monocytogenes was also inactivated usinga double allelic exchange reaction. A pKSV7 plasmid construct containing450-base pair fragments corresponding to the 5′ and 3′ ends of the datgene PCR product, which had been joined together by an appropriate PCRreaction, was introduced into Listeria. A double allelic exchangereaction between the chromosomal dat gene and the dat plasmid constructresulted in the deletion of 30% of the central bases of the dat gene.

Infection of Cells in Culture. To examine the intracellular growth ofthe attenuated strain of Listeria in cell culture, monolayers of J774cells, a murine macrophage-like cell line, primary murine bone marrowmacrophages, and the human HeLa cell line, were grown on glasscoverslips and infected as described (Portnoy et al., 1988, supra). Toenhance the efficiency of infection of HeLa cells, a naturallynon-phagocytic cell line, the added bacteria were centrifuged onto theHeLa cells at 543.times.g for 15 minutes. At various times afterinfection, samples of the cultures were obtained in order to performdifferential staining for the determination of viable intracellularbacteria, or for immunohistochemical analysis.

Immunohistochemistry. Coverslips with attached infected macrophages orHeLa cells were washed with PBS, and the cells were fixed in 3.2%formalin and permeabilized using 0.05% Tween 20. Listeria were detectedusing rabbit anti-Listeria 0 antiserum (Difco Laboratories) followed byLSRSC-labeled donkey anti-rabbit antibodies or coumarin-labeled goatanti-rabbit antibodies. Actin was detected using FITC- or TRITC-labeledphalloidin. To distinguish extracellular (or phagosomal) fromintracytoplasmic bacteria, the former were stained prior topermeabilization of the cells.

Induction of listeriolysin O-specific CTLs. Female BALB/c mice, 6 to 8weeks of age (Charles River Laboratories, Raleigh, N.C.) were immunizedby intraperitoneal inoculation with either wild-type ordal.sup.-dat.sup.-strains of L. monocytogenes. After 14 days, some ofthe mice were boosted with a second inoculation containing the samenumber of microorganisms as were given in the first inoculation. Ten ormore days after the last inoculation, 6.times.10.sup.7 splenocytesobtained from a given animal were incubated in Iscove's modified DMEMwith 3.times.10 splenocytes from that same animal that had been loadedwith 10 .mu.M listeriolysin O (LLO) peptide 91-99 during a 60 minuteincubation at 37.degree. C. After five days of in vitro stimulation, theresulting cultures were assayed for the presence of CTL activity capableof recognizing LLO-peptide-labeled P815 cells following previouslypublished procedures (Wipke et al., 1993, Eur. J. Immunol. 23:2005-2010;Frankel et al., 1995, supra). Every determination of lytic activity wascorrected for activity in unlabeled target cells, which exhibitedbetween 1 and 10 percent lysis.

Animal protection studies. Female BALB/c mice (Bantin-Klingman,Freemont, Calif.) at 8 weeks of age were immunized with approximately0.1 LD.sub.50 of viable wild-type L. monocytogenes or thedal.sup.-dat.sup.-0 double mutant strain in 0.2 ml of vehicle, by tailvein injection. Three to four weeks following immunization, groups offour to five mice each were challenged with approximately 10 LD.sub.50of viable wild-type L. monocytogenes strain 10403 in 0.2 ml of vehicle,by tail vein injection. Spleens were removed from the mice 48 hourslater and were homogenized individually in 4.5 ml PBS-1%proteose-peptone using a tissue homogenizer (Tekmar). The homogenateswere serially diluted and plated onto BHI agar. Log.sub.10 protectionwas determined by subtracting the mean of the log.sub.10 CFU/spleenvalues of the test group from the mean of the log.sub.10 CFU/spleenvalues of the normal control group.

Construction of an Auxotrophic Attenuated Strain of L. monocytogenesUseful as a Vaccine: Construction of an Attenuated Strain of L.monocytogenes Defective in Cell Wall Synthesis.

L. monocytogenes was examined to determine whether the bacteria harborgenes for the synthesis of D-alanine. The alanine racemase (dal) gene,used by many microorganisms for the synthesis of D-alanine, has beensequenced in Salmonella (Galakatos et al., 1986, Biochemistry25:3255-3260; Wasserman et al., 1984, Biochemistry 23:5182-5187), B.subtilis (Ferrari et al., 1985, Bio/technology 3: 1003-1007), and B.stearothermophilis (Tanizawa et al., 1988, Biochemistry 27:1311-1316),but the gene has not been reported in Listeria. Primers based on thesequences (adjusted for preferred codon usage in Listeria) of two highlyconserved regions of the dal gene in two different gram-positiveorganisms were employed in a PCR reaction performed on L. monocytogeneschromosomal DNA to search for evidence of the dal gene in Listeria. Aproduct that exhibited significant homology with the published dal genesequences was obtained. The sequence of the remainder of the L.monocytogenes dal gene was determined as described herein and isdepicted in FIG. 1. The translated protein sequence is compared withalanine racemases of the other gram-positive organisms in FIG. 2.

The dal gene was inactivated by an in-frame insertion of a 1.35 kbfragment of DNA encoding erythromycin resistance at an Spe1 site nearthe center of the gene. The resulting dal.sup.-bacteria were found togrow both in rich bacteriological medium (BHI) as well as in a syntheticmedium in the presence or absence of D-alanine. Mutation of the dal genewas also achieved by an in-frame deletion covering 82% of the gene withthe same effect.

A second enzyme used by some bacteria for synthesis of D-alanine isD-amino acid aminotransferase, encoded by the dat gene (Tanizawa et al.,1989, J. Biol. Chem. 264:2450-2454; Pucci et al., 1995, J. Bacteriol.177:336-342). Following the same strategy used to detect the dal gene inL. monocytogenes, a PCR product that exhibited significant sequencehomology with known dat genes and gene products was obtained. Thesequence obtained from the PCR product was only the partial genesequence, and remainder of the dat gene gene sequence (as depicted inFIG. 3) was determined according to procedures described herein. Thededuced protein sequence of the L. monocytogenes dat gene is comparedwith other dat gene products in FIG. 4.

The L. monocytogenes dat gene was inactivated by in-frame deletion of31% of its central region. The growth of the resulting dar bacteria invarious bacteriological media was again found to be independent of thepresence of D-alanine.

A double mutant strain of L. monocytogenes, dal.sup.-dat.sup.-, wasproduced by a double allelic exchange reaction between theerythromycin-resistant dal.sup.-organism and the shuttle vector carryingthe dat gene deletion. The growth of the double mutant inbacteriological media was found to be completely dependent on thepresence of D-alanine (FIG. 5). A double mutant containing deletions inboth of the genes, designated dal.sup.-dat.sup.-12, had the samephenotype. The growth of the double-deletion strain in the absence ofD-alanine could be complemented by a plasmid carrying the dal gene of B.subtillis. All of the dal.sup.-dat.sup.-double mutant experimentsreported in the following examples employed the dal.sup.-dat.sup.-1double mutant.

Expression of the Defective Phenotype Following Infection of EukaryoticCells.

To determine whether a defect in the ability of L. monocytogenes tosynthesize D-alanine would be expressed as an inability to replicate inthe cytoplasm of eukaryotic cells because of the absence of the requiredD-alanine in the cytoplasm, several different cell lines and primarycells in culture were infected with the wild-type and mutant strains ofthis organism.

J774 cells are a mouse macrophage-like cell line that readily take up L.monocytogenes by phagocytosis and permit its cytoplasmic growthfollowing escape of the bacteria from the phagolysosome (Tilney et al.,1989, J. Cell Biol. 109:1597-1608).

FIG. 6 depicts typical J774 cells as observed at 5 hours after infectionwith about 5 bacteria per cell of either wild-type Listeria (Panel A) orthe double dal.sup.-dat.sup.-mutant Listeria (Panel B). Whereas largenumbers of bacteria were observed to be associated with mouse cellsinfected with wild-type Listeria, few were seen following infection withthe double mutant bacteria. Infection by double mutant bacteria inculture medium containing D-alainine permitted bacterial growth whichwas indistinguishable from that seen in cells infected with wild typeListeria (FIG. 6, Panel C).

Some J774 cells contained small round darkly-staining objects, often inpairs, that may be spheroblast-like bacteria, although they were notexamined further. When these cells were infected at highermultiplicities (a multiplicity of infection of about 1-10), many cellscontained multiple microorganisms, but the double mutant again failed tomultiply. Most double mutant-infected cells possessed pychnotic nucleiand a pale cytoplasm and presumably were dead; mouse cells harboringwild-type Listeria did not exhibit this property at any time afterinfection.

To quantify some of these observations, the number of intracellularbacteria (defined by gentamicin resistance) that could form colonies onmedium containing D-alanine was enumerated at several times afterinfection (FIG. 7). The data clearly demonstrate that the double mutantwas unable to replicate in J774 cells, and in fact slowly died duringthe course of the experiment. The data also illustrate that thereplication-defective phenotype of the double mutant was suppressed bythe inclusion of D-alanine (at 100 .mu.g/ml) in the tissue culturemedium at the time of infection. This suppression was reversed within 2hours after removal of the D-alanine. The phenotype of the mutantbacteria was also examined in mouse bone marrow-derived macrophages andin the HeLa cell line of human epithelaial cells. It was determined thatthe double mutant was unable to replicate in either of these cell typesas well (FIG. 7, Panels B and C).

It was again observed that double-mutant-infected macrophages possessedpychnotic nuclei more frequently than did macrophages infected withwild-type bacteria. Infection of bone marrow macrophages was employed toexamine the intracytoplasmic status of the bacteria. Within a few hoursafter infection of cells by L. monocytogenes, when the bacteria haveescaped from the phagosome, host actin filaments form a dense cloudaround the intracytoplasmic bacteria, and then rearrange to form apolarized comet tail which propels the bacteria through the cytoplasm(Tilney et al., 1989, supra). The actin can readily be visualized usingappropriately labeled anti-Listeria antibodies. At 2 hourspost-infection using a multiplicity of infection of about 5 bacteria percell, 25% of wild type bacteria associated with J774 macrophages weresurrounded with a halo of stained actin (FIG. 8, Panel A), and at 5hours, virtually 100% of infected cells exhibited actin staining, somecells having long actin tails (FIG. 8, Panel B). However, the stainingof actin in double-mutant infected macrophages was much rarer (less than2%) when compared with wild type infected cells. Nevertheless, ifD-alanine was present during only the 30 minute period of bacterialadsorption, at 2 hours post-infection 22% of the mutant cell-associatedbacteria were surrounded with actin (FIG. 8, Panel C); at 5 hours, thisnumber of intracytoplasmic bacteria had risen to only 27% (FIG. 8, PanelD). If D-alanine was present during the entire infection period (FIG. 8,Panel E), the result observed in these cells at 5 hours wasindistinguishable from those observed in wild type infected cells.

Since J774 cells have long been culture adapted and reflect very few ofthe normal properties of tissue macrophages, the entry of mutantbacteria into the cytosol of primary bone marrow macrophages which hadbeen in culture for only 6 days was examined. Because these cellsdemonstrate the high bacterial killing capacity of normal macrophages,they were infected at a ratio of about 50 bacteria per cell. Under theseconditions, at 2 hours after infection, 6.8% of the double mutantbacteria were found to be associated with actin in these cells, and thisnumber increased to the same level as that observed after wild typeinfection (19%) by the inclusion of D-alanine for the first 30 minutesof the infection (18.2%) or for the entire period of infection (19.4%).Therefore, depending on the cell type examined, mutant bacteria in theabsence of D-alanine either exhibited a very low or moderate efficiencyof entering the host cytosol, or exhibited reduced binding of actin ontotheir surface. However, the brief presence of D-alanine during theinitial phase of infection allowed a normal fraction of bacteria toenter the cytosol and bind actin.

Induction of an Immune Response Using the Attenuated Bacteria.

Infection of mice with L. monocytogenes produces a long-lived state ofspecific immunologic memory that enables the infected host to resistlethal challenge by the same organism for months following the primaryinfection. To determine whether infection of mice with sub-lethal dosesof the double mutant could induce this important long lasting state ofimmunity, the following experiments were performed.

Mice were injected intravenously with 2.times.10.sup.7 (<0.05 LD.sub.50)of the double mutant and were challenged 3 to 4 weeks later with 10LD.sub.50 of wild type L. monocytogenes. D-alanine (20 mg) was providedin the initial inoculum of mutant organisms to be certain that theorganisms were fully viable at the time of initial infection (this hadthe effect of reducing the LD.sub.50 about 10 fold). The data presentedin FIG. 9 demonstrate that the level of antilisterial protection wasapproximately 3 log.sub.10 following a single infection by the mutantbacteria, a similar level of protection to that generated byimmunization with the wild-type organism. The same dose of mutantbacteria injected without D-alanine provided little protection.

To determine whether the high degree of protection generated by themutant bacteria could be accounted for by their survival and replicationin the infected mice, the spleens of infected animals were removed andthe number of surviving mutant bacteria was assessed. In FIG. 10 thereis shown evidence which indicates that in the absence of D-alanine, fewmutant organisms survived for more than one day after infection; thepresence of D-alanine in the initial inoculum permitted a few bacteriato survive longer. Importantly, the almost complete protection obtainedusing mutant bacteria occurred in spite of the fact that by 2 dayspost-infection more than 100-fold fewer bacteria were detected in thespleens of mutant infected mice compared with wild type infectedanimals.

Listerolysin O peptide 91-99 is the major epitope of the listerolysin Oprotein and one of the major epitopes to which mice respond whenmounting a cell mediated immune response against infection with L.monocytogenes (Bouwer et al., 1996, Infect. Immun. 64:2515-2522; Hartyet al., 1992, J. Exp. Med. 175:1531-1538; Pamer et al., 1991, Nature353:852-855). To determine whether the protective immunity generated byinfection with the dal.sup.-dat.sup.-double mutant strain of L.monocytogenes was associated with the induction of cytolytic T cells,splenocytes obtained from infected animals were assayed for theirability to lyse target cells loaded with this peptide. In FIG. 11 thereis shown the fact that animals that were infected intraperitoneally with3.times.10.sup.7 and were provided D-alanine subcutaneously both beforeand after infection exhibited strong CTL responses directed against theLLO peptide. Likewise, mice provided with D-alanine in their drinkingWater before and after infection mounted a modest CTL response aftersingle infection with 3.times.10.sup.7 mutant bacteria. In the absenceof D-alanine, animals infected with and boosted one time with3.times.10.sup.7 bacteria, also exhibited a modest CTL response to LLOpeptide 91-99. Single infection with 3.times.10.sup.7 of the doublemutant bacteria in the absence of D-alanine produced no significantresponse (FIG. 11).

The disclosures of each and every publication, patent, and patentapplication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of inducing an immune response against a cancer cell in amammal, the method comprising administering to said mammal anauxotrophic attenuated strain of Listeria comprising an antigenexpressed by said cancer cell.
 2. The method of claim 1, wherein theauxotrophic attenuated strain of Listeria comprises a mutation in thedal gene.
 3. The method of claim 1, wherein the auxotrophic attenuatedstrain of Listeria comprises a mutation in the dat gene.
 4. The methodof claim 3, wherein the auxotrophic attenuated strain of Listeriafurther comprises a mutation in the dal gene.
 5. The method of claim 1,wherein said antigen is expressed from a vector.
 6. The method of claim1, wherein said antigen is expressed from the Listeria genome.
 7. Themethod of claim 1, wherein said auxotrophic attenuated strain ofListeria is administered orally, parenterally, intranasally,intramuscularly, intravascularly, intravenously, intrarectally, orintraperitoneally.
 8. The method of claim 1, wherein said cancer cell isa cervical cancer cell.
 9. The method of claim 1, wherein said cancercell is a melanoma cancer cell, a breast cancer cell, or a leukemiacell.
 10. The method of claim 1, wherein said Listeria is L.monocytogenes.
 11. The method of claim 26, wherein said antigen is HPVE6, HPV E7, or a combination thereof.
 12. The method of claim 1, whereinsaid antigen is the bcr/abl antigen.
 13. The method of claim 1, whereinsaid antigen is MAGE1, MZ2-E or a combination thereof.
 14. The method ofclaim 1, wherein said antigen is MVC-1, HER-2 or a combination thereof.15. The method of claim 1, wherein said antigen is expressed as a fusionprotein with listeriolysin O (LLO), phosphatidylinositol-specificphospholipase (PI-PLC), or a combination thereof.
 16. The method ofclaim 1, wherein said antigen is expressed from a Listeria hly, p60,actA, picA, mpl, plcB, or inlA gene promoter.
 17. The method of claim 1,wherein said immune response is a cytotoxic T cell response.